The present invention relates to a multi-purpose vector. The vector can be for expressing at least one heterologous protein in a suitable cell such as E. coli or other Gram negative bacteria. More specifically, the present invention relates to: a vector for expression of heterologous proteins comprising nucleic acid molecules for: an origin of replication region, optionally but preferably a selection marker (which can be a coding nucleic acid molecule inserted in a restriction site), a promoter, an initiation region e.g. a translation initiation region and/or a ribosome binding site, at least one restriction site and preferably multiple restriction sites, and a transcription terminator; a method for extracting recombinant protein without lysing the cell, e.g., bacteria; and a method for purifying isolated recombinant protein. The vector can facilitate the thermo-regulated production of a heterologous protein or proteins, e.g., pro-insulin.
Several publications are referenced in this application. Full citation to these publications is found at the end of the specification, immediately preceding the claims, or where the publication is mentioned; and each of these publications is hereby incorporated by reference. These publications relate to the state of the art to which the invention pertains; however, there is no admission that any of these publications is indeed prior art.
Recombinant DNA technology has enabled the expression of foreign (heterologous) proteins in microbial and other host cells. A vector containing genetic material directing the host cell to produce a protein encoded by a portion of the heterologous DNA sequence is introduced into the host, and the transformant host cells can be fermented and subjected to conditions which facilitate the expression of the heterologous DNA, leading to the formation of large quantities of the desired protein.
The advantages of using a recombinantly produced protein in lieu of isolation from a natural source include: the ready availability of raw material; high expression levels, which is especially useful for proteins of low natural abundance; the ease with which a normally intracellular protein can be excreted into the expression medium, facilitating the purification process; and the relative ease with which modified (fusion) proteins can be created to further simplify the purification of the resultant protein.
However, the aforementioned benefits of recombinant DNA technology are also accompanied by several disadvantages, namely: the required elements of the active protein which result from post-translational modification (i.e., glycosylation) may not be carried out in the expression medium; proteolytic degradation of newly formed protein may result upon expression in host cells; and the formation of high molecular weight aggregates, often referred to as xe2x80x9cinclusion bodiesxe2x80x9d or xe2x80x9crefractile bodiesxe2x80x9d, which result from the inability of the expressed proteins to fold correctly in an unnatural cellular environment. The recombinant protein cannot be excreted into the culture media upon formation of inclusion bodies.
Inclusion bodies contain protein in a stable non-native conformation; or, the protein aggregates may be amorphous, comprised of partially and completely denatured proteins, in addition to aberrant proteins synthesized as a result of inaccurate translation. Such inclusion bodies constitute a large portion of the total cell protein.
Inclusion bodies present significant problems during the purification of recombinant proteins, as they are relatively insoluble in aqueous buffers. Denaturants and detergents, i.e., guanidine hydrochloride, urea, sodium dodecylsulfate (SDS) and Triton X-100, may be necessary to isolate the proteins from the inclusion bodies, often at the expense of the biological activity of the protein itself, resulting from incorrect folding and modification of the amino acid residues in the sequence.
Additionally, a result of the expression of recombinant DNA in E. coli is the accumulation of high concentrations of acetate in the media, mainly during the induction phase. The deleterious effect of acetate accumulation (greater than 5 g/L) on cell growth and recombinant protein expression has been well documented in the literature.
Further, the recovery of the desired protein from inclusion bodies is often complicated by the need to separate the desired protein from other host cellular materials, in addition to separating the desired protein from inclusion body heterologous protein contaminants. The latter problem results from the strong attraction that inclusion body proteins have for one another, due to strong ionic and hydrophobic interactions.
Consequently, most established protocols for the isolation of recombinant proteins from inclusion bodies result in large quantities of biologically inactive material, and very low yields of active protein, uncontaminated by extraneous heterologous protein.
Researchers have focused on the manipulation of phage in order to stimulate protein synthesis by a variety of methods.
The promoters of the Lambda phage (PL and PR) are strong promoters that are negatively controlled by the repressor coded by the gene cl. The mutation cl857 rendered the repressor inactivate at temperatures above 37xc2x0 C. Thus, the expression of a sequence controlled by these promoters and by the repressor cl857 can be activated by a simple change in temperature. These promoters are often used in E. coli expression vectors, because they are strong and efficiently repressed (Denhardt and Colasanti, 1987).
Remaut et al. (1981) constructed a set of plasmids containing the promoter PL. The promoter and the trp region of the gene were taken from a family of phages (trp44) and inserted in the plasmid pBR322, creating the first plasmid of a series, plasmid pPLa2. After several manipulations, other plasmids were obtained. The plasmids pPLa2 and pPLa8 contained the promoter PL fragment, the origin of replication, the ampicillin resistance gene from the plasmid pBR322, and a kanamycin resistance gene from the plasmid pMK20. The promoter region contained the promoter/operator and the nutL site (antitermination), but it was lacking the beginning of the gene N.
The plasmids pPLc236, pPLc28 and pPLc24 are different from the previously identified plasmids, with respect to the direction of transcription from the promoter PL in relation to the orientation of the origin of replication, as found in pBR322 (a=anticlockwise, c=clockwise). The kanamycin resistance gene is absent in these three vectors. The difference between pPLc236 and pPLc8 is the presence or absence of a region (present in the former and absent in the latter), which affects the region of unique cloning sites. pPLc24 was derived from pPLc28 by insertion of a region containing the ribosome binding site of the gene for replicase from the phage MS2, enabling the expression of eukaryotic genes.
These plasmids were tested with the expression of different genes, e.g., the gene trpA from Salmonella typhimurium, cloned in the plasmid pPLc23 (predecessor of pPLc236), which showed 40% induction of product in relation to the total cellular protein. pLc236 programmed in E. coli resulted in a expression of the gene ROP as 20% of the total protein (Muesing et al., 1984). The proteins p4 and p3 of the phage 29 of Bacillus subtilis, were also produced from pPLci and reached 30% and 6%, respectively, of the total cellular protein induced in E. coli, after thermal induction (Mellado and Salas, 1982).
In 1983, Remault et al. (1983a) built a plasmid pPLc245, derived from pPLc24, in which the initial coding region of replicase was deleted and a region with several unique cloning-sites was added, permitting direct expression. The gene for human xcex1-interferon was cloned into this plasmid, resulting in induction of protein of approximately 2% to 4% of the total cellular protein. For xcex1-interferon, the levels of expression varied from 3% to 25% of the cellular protein, depending on the plasmids used, e.g., pPLc245, pPLc28 and pCP40, and on the presence of a transcription-terminator from phage T4 (Simons et al., 1984). The plasmid pCP40, derived from pPL, was built by Remaut et al. (1983b). The promoter-region was transferred to a plasmid derived from pKN402 with temperature dependent xe2x80x98runawayxe2x80x99 replication. When the cultures are heated to 42xc2x0 C, the repressor cl857 is deactivated and the promoter PL is liberated, resulting in an increase in the number of copies of the plasmid pCP40, by approximately ten fold.
Crowl et al. (1985) relates to four plasmids containing the promoter PL. The plasmid pRC23 was built containing the promoter PL and a synthetic Shine-Dalgarno region, without the codon ATG, cloned in the plasmid pRC2 (derived from pBR322). To build the other three plasmids, pEV-vrf1, pEV-vrf2 and pEV-vrf3, a region with unique cloning sites was inserted, adding the initial ATG codon, such that in each one the reading frames are on phase. The plasmid pRC23 was used for the expression of interleucine-2 and xcex1-interferon, with a level of 10% to 20% of the total cellular protein.
Lautenberg et al. (1983) built the plasmid pJL6, containing the promoter PL, which codes for initiation of translation of the gene cII of the phage, with unique ClaI and HindIII cloning sites, located at 50 bp from the initial ATG site. Genes, adequately cloned in these sites are induced, producing fusion proteins with the protein CII. Seth et al. (1986) modified this vector so that the induction of proteins could occur without fusion. Three plasmids were constructed, containing a KpnI site in pANK-12, an HpaI site in pANH-1, and an NdeI site in pPL2 of the initial codon ATG of the gene CII of pJL6. In pANH-1, the amino acid xe2x80x98valinexe2x80x99 occurred more frequently in the amino-end of the induced protein. Production of oncogenes was obtained from these vectors. Chang and Peterson (1992) also modified the plasmid pJL6 and built a line of plasmids, pXC, in which the region for initiation of translation of the gene CII was substituted by a synthetic one. Additionally, a region was inserted having several unique cloning sites. The region CII affects the efficiency of the translation if the expression is required without fusion. With the synthetic region, the efficiency rose between 10 and 20 times, depending on the spacing region between SD and ATG. The expression reached 48% of the total cellular protein for the protein 14-3-3 of cow brain, of which the DNA had been amplified by PCR.
Schauder et al. (1987) built a line of plasmids derived from pJL6, containing the promoters PR and PL in tandem, the region SD of the gene atpE (for subunit of ATPase), with the transcription terminator of the bacteriophage fd and with the gene of the repressor cl857. These plasmids were named pJLA501 to xe2x88x9205 and differ in the regions of the multiple cloning sites. On testing the expression of the gene atpA (for a subunit of ATPase), an induction of 50% of the total cellular protein was found. The genes sucC and sucD, respectively, showed 30% and 15% induced protein in relation to the total cellular protein.
Rosenberg et al. (1983) built the plasmid pKC30 and its derivatives. The vector pKC30 is used for the expression of bacterial genes containing their proper translation-regulation regions. This vector contains a unique cloning HpaI site, located 321 bp downstream from the promoter PL, within the coding region of the gene N. The expression of the activator CII and eight mutants in just one amino acid was achieved in the vector pKC30. Because CII is quickly recycled in E. coli and deleterious for cell growth, with insertion and expression of its gene in pKC30, levels of 3% to 5% of the total cellular protein were reached. The production of the protein CII rose when the protein N (anti-terminator) was provided by the host-cell, because of the presence of the xe2x80x98upstreamxe2x80x99 sequences of the gene CII of the sites nutL, nutR (for anti-termination) and tr1 (for termination). Other proteins were expressed from pKC30, such as the protein B of the phage Mu (Chaconas et al., 1985) and the protein UvrA of E. coli expressed at levels of 15% and 7% of the total cellular protein respectively.
For the expression of eukaryotic genes the plasmid pAS1, derived from pKC30, was built with the cloned gene CII. The complete coding region of CII was deleted and a BamHI site was added immediately xe2x80x98downstreamxe2x80x99 of the ATG initiation codon. In this manner, the regulating regions for translation were maintained in the vector and a eukaryotic or synthetic gene can be expressed if cloned correctly to the BamHI site. Expression of the gene for the antigen t of the virus SV40 resulted in this vector in levels of 10% of the total cellular proteins, after one hour of thermal induction (Rosenberg et al., 1983). Lowman et al. (1988) modified the plasmid pAS1, introducing a NcoI site in the initial ATG, creating the plasmid named pAS1-N. Expression of the gene CAT and fusion with proteins of the virus SV40 were obtained. Later, Lowman and Bina (1990) used these products to study the effect of temperature in thermal induction.
Mott et al. (1985) used pKC30 and pAS1 to express the bacterial gene rho and verified that the thermal induction did not result in high levels of expression of the protein Rho. Induction with nalidixic acid and mitomicina C was tested in the host cI, which provoked the induction of the syntheses of Rec a, resulting in an inactivation of the repressor cI. In this manner, levels of expression varying from 5% to 40% of the cellular protein were reached.
Hence, the manipulation of plasmids for expression of a protein or peptide of interest is a developing area and a method for the induction of complex proteins such as pro-insulin via manipulation of a plasmid and a plasmid therefrom, have not heretofore been developed or suggested.
U.S. Pat. No. 4,734,362, to Hung et al., is directed to a method of isolating polypeptides produced recombinantly in inclusion bodies. The disclosed method includes the cell lysis, and recovery of inclusion bodies comprising the desired recombinant protein, solubilization with denaturant, protection of the sulfhydryl groups of the recombinant protein, derivatization of cationic amino groups of the protein, and recovery of the derivatized recombinant protein.
Olson, U.S. Pat. No. 4,518,526 relates to a method of releasing active proteins from inclusion bodies by cell lysis, centrifugation, denaturation and renaturation. The patent teaches the necessity of the disruption of the cell to separate the soluble and insoluble protein, followed by treatment of the insoluble fraction with a strong denaturant, and recovery of the renatured heterologous protein.
Rausch, U.S. Pat. No. 4,766,224 is directed to a method of purification and solubilization of proteins produced in transformed microorganisms as inclusion bodies. The purification is effected by solubilization of the inclusion bodies in detergent, treatment with a strong denaturant, followed by chromatographic separation to obtain renatured active protein.
Builder et al., U.S. Pat. No. 4,620,948 is concerned with a process for isolating and purifying inclusion bodies by lysing the cell culture, precipitation of protein, denaturation of the insoluble fraction, and renaturation to isolate the refractile protein.
Similarly, U.S. Pat. Nos. 4,734,368, 4,659,568, 4,902,783, 5,215,896, and EP 337,243 and WO 87/02673 are each directed to methods of purifying proteins entrapped in inclusion bodies. These methods use of the following techniques (alone or in combination): cell lysis, denaturation, chromatographic separation, centrifugation, manipulation of the denaturation/renaturation of the protein, and the attachment of leader peptides which facilitate the separation of the proteins from the inclusion bodies.
Each of the aforementioned prior art processes utilize methods which disrupt the cell to release the inclusion bodies from the cellular material. There is no teaching or suggestion of a means for isolating inclusion bodies from cellular material without the disruption of the cell, nor is there a motivation to derive such a method from the teachings of the prior art. However, the lysis or disruption of cells is disadvantageous as it allows contaminants to be present with the desired protein, such as lipopolysaccharides, which are very difficult to separate from the desired protein.
U.S. Pat. Nos. 4,877,830, 5,115,102, 5,310,663, and EP 656,419, WO 91/11454, WO 91/16912, WO 94/07912, Proc. Natl. Acad. Sci. (1991) 88 (20), and Mol. Biol. Rep. (1993) 18: 223-230 are each directed to affinity purification of proteins. These documents relate to the use of (alone or in combination): metal chelate affinity chromatography for chromatographic separation of proteins having neighboring histidine residues, immunoaffinity chromatography, and the use of amino acid mimetics as eluents in affinity purification of proteins.
U.S. Pat. Nos. 4,766,205, 4,599,197, 4,923,967, and EP 312,358, EP 302,469, Biochemistry (1968), 7 (12), 4247, and J. Biological Chemistry (1959), 234 (7), 1733 are each directed to methods of sulfitolysis, i.e., the treatment of a protein, solubilized in a strongly denaturing solution, with a mild oxidant in the presence of sulfite ion, which converts cysteine and cystine residues to protein-S-sulfonates. The strongly denaturing solution is weakened to permit refolding, and disulfide linkages are reformed using a sulfhydryl compound, in the presence of the corresponding disulfide (oxidized) form. Similarly, EP 208,539 and WO 87/02985 are directed to methods of facilitating protein refolding in vitro.
EP 264,250, GB 2,067574, EP 055,945, MMW (1983) 125 (52), 14, J. Biol. Chem. (1971) 246 (22), 6786-91, J. Chrom. (1989) 461: 45-61 are each directed to insulin, its production from pro-insulin, and the purification of insulin and pro-insulin.
U.S. Pat. No. 4,578,355, to Rosenberg, is directed to the derivation and use of the PL transcription unit. EP 363,896 is directed to the use of ultrafiltration in protein purification.
Human insulin, a proteolytic digestion product of pro-insulin, is a polypeptide hormone produced by beta cells of the islets of Langerhans in the pancreas. Its purpose is to decrease the amount of glucose in the blood by promoting glucose uptake by cells, and increasing the capacity of the liver to synthesize glycogen. The action of insulin is antagonistic to glucagon, adrenal glucocorticoids and adrenaline, and its deficiency or reduced activity produces diabetes with a raised blood sugar level.
Human insulin has been prepared from several sources, including: isolation from human pancreas, peptide synthesis, the semisynthetic conversion from porcine insulin and fermentation of E. coli bacteria or Saccharomyces cerevisiae yeast, suitably encoded by DNA recombinant methods. These methods suffer from poor yield and cost efficiency, and the development of a high yielding, cost effective method of producing human insulin for the treatment of diabetes has been the subject of much research efforts in recent years.
Hence, a method for the induction of human pro-insulin via recombinant techniques has not heretofore been realized, wherein the protein may be isolated in substantial quantities from inclusion bodies, especially such a method wherein cell lysis or cell disruption is avoided.
Objects of the present invention may include providing at least one of: a vector comprising at least one nucleic acid such as DNA for cloning of a nucleic acid or for expression of at least one heterologous protein by a cell such as Gram negative bacteria (the vector can comprise a nucleic acid molecule, e.g., DNA, encoding: an origin of replication region, optionally and preferably a selection marker (which can be a coding nucleic acid in a restriction site), a promoter, an initiation region e.g. a translation initiation region and/or a ribosome binding site, at least one restriction site for insertion of heterologous nucleic acid, e.g., DNA, encoding the heterologous protein, and a transcription terminator); a method for extracting a recombinant protein from within a cell such as a recombinant Gram negative bacteria having a cell membrane; and, a method for purifying an isolated recombinant human insulin.
Accordingly, the present invention provides a vector comprising at least one nucleic acid molecule such as DNA for cloning of a nucleic acid molecule, or more preferably, for expression of at least one heterologous protein by cell such as a Gram negative bacteria. The vector can comprise DNA encoding the following: an origin of replication region, optionally and preferably a selection marker (which can be coding DNA in a restriction site), a promoter, an initiation region e.g. a translation initiation region and/or a ribosome binding site, at least one restriction site for insertion of heterologous DNA encoding the heterologous protein, and a transcription terminator.
The Gram negative bacteria can be E. coli. The origin of replication region can be from plasmid pUC8. The initiation region can be a translation initiation region and can be synthetic, e.g., synthetic Shine-Dalgarno regions from gene 10 of phage T7. The selection marker can be a tetracycline resistance marker. Alternatively or additionally, selection of transformed cells containing the vector can be on the basis of a product expressed by the heterologous DNA encoding the heterologous protein. The promoter can be a PL promoter. And, the transcription terminator can be a Rho-independent one.
Thus, the invention can provide a vector comprising DNA for expression of a heterologous protein by a Gram negative bacteria. The vector can comprise at least one nucleic acid molecule, e.g., DNA, encoding the following: an origin of replication region, a selection marker, a promoter, a translation initiation region or a ribosome binding site, at least one restriction site for insertion of heterologous DNA encoding the heterologous protein, and a transcription terminator.
The DNA encoding the at least one restriction site preferably encodes multiple restriction sites; and, the multiple restriction sites are preferably Ncol, EcoRI, StuI, PstI, BamHI, and BspEI.
The present invention further provides a vector for expression of a pro-insulin by a Gram negative bacteria. That is, in the inventive vector, at the at least one restriction site for insertion of heterologous DNA (or a heterologous nucleic acid sequence) there can be inserted a nucleic acid molecule such as DNA encoding pro-insulin, e.g., human pro-insulin. The protein expressed by the inserted nucleic acid molecule, e.g., pro-insulin such as human pro-insulin, can contain tag or a marker, for instance, a His tag (which is useful for separating, isolating and/or purifying the protein).
And therefore, more generally, inventive vectors can include at least one exogenous coding nucleic acid molecule at the at least one restriction site for insertion of a heterologous nucleic acid molecule, e.g., exogenous coding DNA can be at the at least one restriction site for insertion of heterologous DNA. Further, the exogenous coding DNA can encode, in addition to the heterologous protein, a marker or tag, for instance a His tag.
Still further, the invention provides a method for extracting a recombinant protein such as pro-insulin, e.g., human pro-insulin, from within a cell such as a recombinant Gram negative bacteria having a cell membrane, without lysing the bacteria. The method can comprises the steps of:
(a) permeabilizing the cell membrane by contacting the bacteria with a detergent under conditions which facilitate the extraction of native cell proteins from the cell membrane without extracting the recombinant protein from the cell membrane;
(b) solubilizing the recombinant protein and cell membrane; and
(c) separating the recombinant protein from the cell membrane.
The invention also provides a method for purifying an isolated recombinant protein such as pro-insulin, e.g., human insulin, comprising:
(a) subjecting the isolated recombinant human insulin to sulfitolysis and separating a liquid product therefrom,
(b) subjecting the liquid product from (a) to a Ni-chelating column and obtaining an eluate,
(c) renaturing the eluate from (b),
(d) converting the product from (c) e.g., with trypsin and carboxypeptidase B, and
(e) subjecting the product from (d) to purification, e.g., chromatography, to obtain purified isolated recombinant human insulin.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.